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Abstract:

Disclosed in this specification is a method to promote the growth of
CD4+CD25Foxp3+ nTreg cells in a culture while minimizing the production
of IL-17. The resulting cells are useful in the treatment of
immune-related diseases.

2. The process as recited in claim 1, wherein the IL-17 inhibitor is an
anti-IL-6 antibody.

3. The process as recited in claim 1, wherein the IL-17 inhibitor is an
anti-IL-12 antibody.

4. The process as recited in claim 1, wherein the IL-17 inhibitor is an
anti-IL-23 antibody.

5. The process as recited in claim 1, wherein the IL-17 inhibitor is
IL-27.

6. The process as recited in claim 1, wherein the IL-17 inhibitor includes
at least two compounds selected from the group consisting of an anti-IL-6
antibody, an anti-IL-12 antibody, an anti-IL-23 antibody, and IL-27, and
combinations thereof.

7. A process for producing CD4+CD25+ nTreg cells comprising the step of
expanding the population of the enriched CD4+CD25+ regulatory T cells
while treating the sample with an IL-17 inhibitor.

8. A process for producing CD4+CD25+ nTreg cells comprising the steps
of:expanding the population of the enriched CD4+CD25+ regulatory T cells
while treating the sample with an IL-17 inhibitor; andadministering a
portion of the expanded CD4+CD25+ regulatory T cells to a human being to
treat graft versus host disease.

9. The process as recited in claim 8, wherein the IL-17 inhibitor is an
anti-IL-6 antibody.

10. The process as recited in claim 8, wherein the IL-17 inhibitor is an
anti-IL-12 antibody.

11. The process as recited in claim 8, wherein the IL-17 inhibitor is an
anti-IL-23 antibody.

12. The process as recited in claim 8, wherein the IL-17 inhibitor is
IL-27.

13. The process as recited in claim 8, wherein the IL-17 inhibitor
includes at least two compounds selected from the group consisting of an
anti-IL-6 antibody, an anti-IL-12 antibody, an anti-IL-23 antibody,
IL-27, and combinations thereof.

[0002]This invention relates, in one embodiment, to a method for
minimizing IL-17 production in cell populations during the expansion of
nTreg cells. The resulting nTreg cells are particularly useful for
treating immune-mediated diseases including, but not limited to
autoimmunity, organ transplant rejection and graft versus host disease
(GvHD).

BACKGROUND OF THE INVENTION

[0003]T regulatory (Treg) cells are important in maintaining the
homeostatic balance of the human immune system and immune tolerance. One
of the most well studied types of Treg cells is the naturally occurring
Treg (nTreg) cell (CD4+CD25+Foxp3+ cell). Defects in either the nTreg
cells or in Foxp3 have been linked to unfavorable immune responses such
as autoimmunity, allergic response, and organ rejection. Conversely,
administrations of healthy CD4+CD25+Foxp3+ nTreg cells have demonstrated
therapeutic effects in the treatment of a variety of animal disease
models. Although the nTreg cells are a small fraction of the circulating
lymphocyte pool it has been found that nTreg cells can be expanded ex
vivo to provide clinically useful quantities of nTreg cells. The
possibility therefore exists for using ex vivo expanded nTreg cells to
regulate the immune response of a human being.

[0004]During the process, nTreg cells are withdrawn from peripheral blood
mononuclear cells (PBMC) using magnetic bead-based methods. The enriched
nTreg cells are activated with anti-CD3/CD28 coated beads in the presence
of high concentrations (1000 U/ml) of human recombinant IL-2. Although
the purified cells are enriched for nTreg using the bead-base methods,
the resulting sample is not pure. Due to the lack of nTreg-specific
surface markers, the sample almost always contains non-Treg cells that
expressed similar cell surface markers (e.g. CD4 and CD25). After about
three weeks of culture time, the nTreg cell populations underwent over a
one hundred fold expansion while maintaining their phenotypic expressions
(CD25+ Foxp3+). Careful culturing conditions are needed to prevent the
non-nTreg cells from expanding faster than the nTreg cells and disturbing
the overall composition of the sample. The overgrowth of non-Treg cells
during nTreg expansion not only reduces the potency and effectiveness of
the nTreg cell therapy, but also provides a potential source of
pro-inflammatory T effector cells and cytokines. Thus there is a need to
find strategies and compounds to suppress the activation and growth of
non-Treg cells in the cultured population.

SUMMARY OF THE INVENTION

[0005]Applicants have discovered that human IL17 was produced by a portion
of the non-nTreg cells from ex vivo expanded human nTreg cell pool. Since
the nTreg cells were well known for their immunosuppressive properties,
it was surprising to discover the production of IL-17 in the expansion
culture, which IL17 is known to promote immune mediated diseases.

[0006]IL-17 (JI17A) is a cytokine secreted mainly by activated CD4 and CD8
T cells. In vitro, IL-17 induces the production of several
pro-inflammatory cytokines/chemokines (TNFa, IL-1b, IL-6, IL-8, GM-SCF,
and MCP-1) from various cells types and also may play a role in
neutrophil mediated inflammatory responses. In animal studies, it has
been suggested that IL-17 may be involved in the pathogenesis of
rheumatoid arthritis, osteoarthritis, asthma, inflammatory bowel disease,
and multiple sclerosis. The production of IL-17 is regulated by other
cytokine and co-stimulatory molecules. In mouse system, TGFβ, which
is a key cytokine for the development and functions of certain types of
Treg, is also the key differentiation cytokine for IL-17-producing T
cells (Th17) in the presence of IL-6. Both IL-23 and IL-15 enhances,
while IL-27 reduces, IL-17 production in mouse T cells. Certain
co-stimulatory molecules also play a role in IL-17 production, ICOS
up-regulate while OX40 down-regulate IL-17 production in T cells. Because
of the potential pathological impact of IL-17 in several immune mediated
inflammatory diseases, applicants believe it is desirable to minimize
IL-17 production in cell populations that will be used to treat
inflammatory diseases (for example, CD4+CD25+Foxp3+ nTreg cells).

[0007]Disclosed in this specification are several methods to minimize the
production of IL-17 while expanding a cell population that includes
CD4+CD25+Foxp3+ nTreg cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]The present invention is disclosed with reference to the
accompanying drawing, wherein:

[0009]FIG. 1 is a graphical depiction of the response of a cell culture to
various additives.

[0010]The examples set out herein illustrate several embodiments of the
invention but should not be construed as limiting the scope of the
invention in any manner.

DETAILED DESCRIPTION

[0011]Human natural Treg (nTreg) cells were purified from normal donor
PBMC using the commercially available Miltenyi Treg kit with AutoMacs
(available from Miltenyi). The resulting sample was enriched nTreg cells
relative to the original sample. Similar results were obtained by
depleting CD19+ cells and thereafter performing positive selection of
CD25+ by AutoMacs using Miltenyi CD19 and CD25 beads. Typically, 50-70%
of the enriched cells are Foxp3+ as assessed by intracellular Foxp3
staining and analyzed by flow cytometry. In some studies, nTreg was
enriched through FACS sorting based on CD4+, CD25high and CD127low
population. In this case, the Foxp3+ cells consisted of >90% of the
CD4+CD25+ population.

[0012]The enriched nTreg cells were re-activated with anti-CD3/CD28 coated
beads (Dynal) at a 1:3 cell to bead ratio in X vivo-15 media (Cambrex)
that was supplemented with 10% pooled human serum (Cambrex) in the
presence of 1000 (IU/ml) of human rIL-2 under 37° C. incubation.
In three weeks time, nTreg cells experience over a one hundred fold
expansion. About 50% of the expanded cells were Foxp3+ and the cultured
cells exhibited potent inhibitory activities during in vitro functional
assays. nTreg cells which had been cultured for three weeks were allowed
to rest in IL-2-only media for two days and then re-stimulated with
anti-CD3/CD28 antibodies. Cytokines in the supernatants were measured
with Luminex. Cytokine profiles of the resulting cellular sample, as well
as those from comparable CD25-T cells, were as follows:

[0013]Subsequent studied showed that a minor population of ex vivo
expanded CD4+CD25+ T cells produce IL-17 and about 50% of the
IL-17-producing cells are also Foxp3+. This data was determined as
follows: A population of nTreg cells was purified from normal donor PBMC
either using magnetic beads or through FACS sorting (CD4+CD25highCD127-)
and the starting population of Foxp3+ cells was approximately 60%. Cells
were activated with anti-CD3/CD28 in the presence of IL-2 for three
weeks. The cells were then rested for two days and re-stimulated with PMA
plus ionomycin for five hours and the IL-17 production was detected by
intracellular staining for IL-17 and counter-stained for Foxp3.

[0014]Meanwhile, IL17 Intracellular staining was also performed on the
enriched nTreg prior to expansion of the population. It was determined
that IL17+ cells were present prior to expansion and the majority of the
IL-17+ cells were Foxp3-. Additionally, the production of IL-17 from
CD25- cells was also examined, but no detectable amount of IL-17 was
found.

[0015]These results suggest that CD25+IL-17+(Foxp3+ or Foxp3-) T cells are
present in the PBMC of normal donors. The Foxp3+IL17+ T cells in the
three week nTreg cell samples could have come from the expansion of the
minor population of the starting Foxp3+Il-17+ cells or were converted
from the Foxp3-IL-17+ cells. Since there is a lack of unique cell surface
markers to remove the IL-17+ cells from the nTreg culture, a practical
strategy is to limit the expansion/conversion of the IL-17+ cells in the
nTreg ex vivo culture through the manipulation of culture conditions.

[0016]The production of IL-17 is known to be regulated by other cytokines
including, for example, IL-6, IL-12, IL-23 and IL-27. The phrase "IL-17
inhibitor" refers to any compound capable of reducing the level of IL-17
expression, either directly or indirectly. Several cultures were treated
with IL-17 antagonists. FIG. 1 shows the percentage of IL-17+ cells when
various additives are used.

[0017]While the invention has been described with reference to preferred
embodiments, it will be understood by those skilled in the art that
various changes may be made and equivalents may be substituted for
elements thereof to adapt to particular situations without departing from
the scope of the invention. Therefore, it is intended that the invention
not be limited to the particular embodiments disclosed as the best mode
contemplated for carrying out this invention, but that the invention will
include all embodiments falling within the scope and spirit of the
appended claims.